Primary Sertoli cells cultured in vitro secret a large number of proteins under the influence of either FSH, testosterone, and/or yet to be identified biological factor(s). The Principal Investigator (P.I.) and his colleagues have purified several of these Sertoli cell proteins, and specific antibodies and immunoassays have also been prepared. Many of the Sertoli cell proteins studied in the P.I.'s laboratory during the past grant period can be categorized by their association with: Sertoli cell- germ cell interactions (testins); cell aggregation and regression (clusterin); tissue remodeling (alpha2-macroglobulin, cathepsin L), and metal binding and/or transport (testicular SPARC). The P.I. proposes to use these proteins and other yet to be purified Sertoli cell proteins as markers to study Sertoli cell-germ interactions using an in vitro bicameral culture system which mimics the in vivo physiological condition of testicular cells in the seminiferous epithelium. Preliminary studies have shown that the secretion of alpha2-macroglobulin and clusterin by Sertoli cells is not regulated by either FSH or testosterone; however, germ cell- conditioned media differentially regulate the secretion of these proteins. We will seek to purify the putative germ cell factors that modulate the Sertoli cell secretory function. A sensitive and reliable bioassay using primary Sertoli cell-enriched culture has now been established to monitor the germ cell factors. This bioassay will be used in conjunction with the established protein purification technology established in the P.I.'s laboratory to purify the putative germ cell factors. We propose to purify sufficient quantities of these germ cell factors to allow partial N- terminal amino acid sequence analysis and preparation of monospecific antibodies for physiological studies to define the biochemical relationship between Sertoli cells and germ cells. We also propose to isolate the cDNAs coding for these germ cell factors to study the regulation of their mRNAs under various in vitro and in vivo physiological conditions and for primary sequence determination. The proposed project will provide new tools for studying the physiology of Sertoli cell-germ interactions and the seminiferous epithelium.